Ribonuclease H (RNase H) is an enzyme involved in DNA replication with unique functionality. The enzyme has been shown to be associated with the transcription machinery and to be involved in defining the origin of DNA replication. RNase H has recently been implicated in having an additional DNA proofreading function. The three-dimensional structure and backbone dynamics for unliganded RNase H have been characterized, yet no detailed structural or dynamical information is available for RNase H complexed with substrate or inhibitor. Thus, specific interactions between RNase H and nucleic acids are unavailable and conformational changes in both RNase H and helical DNA/RNA substrate have not been identified. The proposed research will use a combinatorial approach to identify inhibitors specific for RNase H. In addition to the combinatorial approach, site directed mutagenesis will be performed to generate stable substrate/RNase H complexes. The three- dimensional structure and dynamical properties of the RNase H complexes will be characterized using 3D and 4D triple resonance NMR spectroscopic techniques. This research will identify dynamic and structural determinants involved in protein-nucleic acid recognition and enzymatic catalysis.